1. Field of the Invention
The present invention generally relates to nitroderivative compounds such as diazo compounds, and residues thereof (e.g., diazotyrosine-containing proteins). More particularly, the invention relates to such compounds azo-coupled to a signal-producing target compound, and to methods and compositions for identifying or quantifying such compounds, especially in biological specimens. Still more particularly, the invention relates to assessing in vivo nitrative stress, and to aiding in the detection of illness or disease by quantitating or detecting the signal resulting from such azo-coupling.
2. Description of Related Art
The nitration of tyrosine residues in proteins to form 3-nitrotyrosine has been characterized as one of several nitric oxide dependent chemical modifications of proteins. Nitrogen dioxide and derivatives of peroxynitrite (both are products of the non-enzymatic oxidation of nitric oxide in vivo) [1–8], reactive compounds produced from nitrite by peroxidases and other enzymes with metal cofactors, as well as by non-enzymatic Fenton-type reactions [9–14] are main natural protein nitration agents. In addition, it was also shown that intense visible light elevates tyrosine nitration in retina, although the chemistry of this process remains poorly understood [15]. Nitrotyrosine levels increase dramatically when the cells are experiencing nitrosative or oxidative stress, and is associated with inflammation, asthma and atherosclerosis [16, 17]. Nitration of specific residues in proteins may play a role in regulating Alzheimer's and Parkinson disease, and cancer [18–21]. The presence of nitrotyrosine residues in proteins can change their activity and/or half-life and may have physiological effects in the normal conditions. For example, tyrosine nitration of sperm proteins occurs in capacitated human spermatozoa [22]. It was shown that nitrotyrosine residues can lead to other processes of protein modifications [23–25]. One of them is the reduction of nitrotyrosine to aminotyrosine, probably through the nitroso- and hydroxyamino derivatives. This reduction can be both enzymatic and non-enzymatic [26, 27] and is regulated independently on nitration mechanism. Thus, at every moment the cell, organ or organism has a unique composition and pattern of nitro- and aminotyrosine containing proteins.
Formation of aminotyrosine can be attributed to several alternative pathways. Sodum and Fiala found high levels of aminotyrosine (>0.1% of the total protein tyrosine) in the liver of rats treated with 2-nitropropane, a strong hepatocarcinogen, or few other secondary nitroalkanes, oximes as well as hydroxylamine-O-sulfonate [28]. Determination and quantification of these NO-dependent modifications of proteins are necessary to understand the key cellular processes and could have clinical applications [29].